Download and Genetic Classification

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CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
A comprehensive genetic classification of adult acute lymphoblastic leukemia
(ALL): analysis of the GIMEMA 0496 protocol
Marco Mancini, Daniela Scappaticci, Giuseppe Cimino, Mauro Nanni, Valentina Derme, Loredana Elia, Agostino Tafuri, Marco Vignetti,
Antonella Vitale, Antonio Cuneo, Gianluigi Castoldi, Giuseppe Saglio, Fabrizio Pane, Cristina Mecucci, Andrea Camera,
Giorgina Specchia, Alessandra Tedeschi, Francesco Di Raimondo, Giuseppe Fioritoni, Francesco Fabbiano, Filippo Marmont,
Felicetto Ferrara, Nicola Cascavilla, Giuseppe Todeschini, Francesco Nobile, Maria Grazia Kropp, Pietro Leoni,
Antonio Tabilio, Mario Luppi, Luciana Annino, Franco Mandelli, and Robin Foà
The Gruppo Italiano Malattie Ematologiche dell’Adulto (GIMEMA) 0496 protocol, through the central handling of bone
marrow samples at presentation, allowed
us to combine cytogenetic and molecular
information on a large series of adults
with acute lymphoblastic leukemia (ALL)
treated homogeneously, enabling us to
define as broadly as possible their genetic profile and to determine the impact
on outcome of the cytogenetic-molecular
signature. Of 414 patients centrally processed, 325 were considered for the categorization into the following cytogeneticmolecular subgroups: normal, t(9;22)/
BCR-ABL, t(4;11)/MLL-AF4, t(1;19)/E2APBX1, 9p/p15-p16 deletions, 6q deletions,
miscellaneous structural abnormalities,
and hyperdiploid. The inclusion into each
subgroup was based on a hierarchical
approach: molecular abnormalities with
adverse prognosis had precedence over
karyotypic changes with less-defined
prognosis and the latter over ploidy. Patients without abnormalities and those
with isolated 9p/p15-p16 deletions showed
a relatively favorable outcome (median
disease-free survival [DFS], > 3 years).
The t(9;22)/BCR-ABL, t(4;11)/MLL-AF4, t(1;
19)/E2A-PBX1 defined a group with dis-
mal prognosis (median DFS, 7 months),
whereas 6q deletions, miscellaneous aberrations, and hyperdiploidy predicted an
intermediate prognosis (median DFS, 19
months). This study highlights the importance of a combined cytogenetic-molecular profiling of adult ALL at presentation
as a critical independent determinant of
their outcome, providing further evidence
of the necessity of a risk-adapted therapeutic algorithm for an optimal management of these patients. (Blood. 2005;105:
3434-3441)
© 2005 by The American Society of Hematology
Introduction
The analysis of the chromosome and genetic abnormalities that occur in
acute leukemias has provided relevant insights into the prognostic
significance of these genetic lesions, which, in turn, become of potential
importance for the design of risk-adapted therapeutic strategies for
newly diagnosed cases. In the last decade, only a few studies have
analyzed the prognostic value of cytogenetic abnormalities in large
series of adult patients with acute lymphoblastic leukemia (ALL).1-5
Although there is general agreement on the unfavorable clinical
outcome of patients with ALL with t(9;22) or t(4;11) abnormalities, the
prognostic significance of other cytogenetic abnormalities is still debated. This may be attributed to the well-known technical difficulties
associated with chromosome analysis in ALL, the frequent overlap of
numerical and structural changes confounding the prognostic
significance of the single anomaly, and the low frequency of certain
karyotypic changes.6,7 In addition, it should be considered that
most studies have focused on patients undergoing different treatment modalities.1,3,5
Over the last 2 decades, it has become evident that molecular
investigations are a useful integration of cytogenetic analysis,
allowing the detection of molecular alterations in cases with
apparently normal or failed karyotype.8,9 Therefore, an integrated
cytogenetic-molecular approach seems the most appropriate tool
for an accurate genetic characterization of patients with ALL at the
onset of the disease.
From the Department of Cellular Biotechnologies and Hematology, University
“La Sapienza” Rome, Italy; Dipartimento di Scienze Biomediche e Terapie
Avanzate, Sezione di Ematologia, Università di Ferrara, Ferrara, Italy; Division
of Hematology, Department of Clinical and Biological Sciences, University of
Turin, Turin, Italy; CEINGE Biotecnologie Avanzate, Department of
Biochemistry and Medical Biotechnology, Federico II University, Naples, Italy;
Hematology and Bone Marrow Transplantation Unit, University of Perugia,
Perugia, Italy; Hematology, Federico II University, Naples, Italy; Department of
Hematology, University of Bari, Bari, Italy; Hematology, Niguarda Cà Granda
Hospital, Milan, Italy; Department of Medical Sciences, University of Catania,
Catania, Italy; Hematology, Civil Hospital, Pescara, Italy; Hematology, Hospital
Cervello, Palermo, Italy; Department of Medicine, Hospital S. Giovanni
Battista, Torino, Italy; Hematology, Cardarelli Hospital, Naples, Italy;
Hematology, Casa Sollievo della Sofferenza Hospital, San Giovanni Rotondo,
Italy; Department of Clinical and Experimental Medicine, University of Verona,
Italy; Divisione di Ematologia Azienda Ospedaliera, Reggio Calabria, Italy;
Hematology, Azienda Ospedaliera A. Pugliese, Catanzaro, Italy; Hematology,
University of Ancona, Ancona, Italy; Department of Oncology and Hematology,
University of Modena and Reggio Emilia, Modena, Italy; and Azienda
Ospedaliera S. Giovanni Addolorata, Roma, Italy.
Submitted June 28, 2004; accepted December 24, 2004. Prepublished online
as Blood First Edition Paper, January 13, 2005; DOI 10.1182/blood-2004-072922.
3434
Supported by the Associazione Italiana contro le leucemie–Sezione di Roma
(Romail); the Consiglio Nazionale delle Ricerche (CNR), Ministero
dell’istruzione, dell’Università e della Ricerca (MIUR), and Associazione
Italiana Ricerca sul Cancro (AIRC; C.M.); Progetto Finalizzato Oncologia,
Fondazione Internazionale di Ricerca in Medicina (FIRMS); and the Istituto
Superiore di Sanità (R.F.).
A complete list of the members of the Gruppo Italiano Malattie EMatologiche
dell’ Adulto (GIMEMA) appears in the “Appendix.”
Reprints: Marco Mancini, Department of Cellular Biotechnologies and
Hematology, University “La Sapienza” Via Benevento 6 Rome, 00161 Italy;
e-mail: [email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2005 by The American Society of Hematology
BLOOD, 1 MAY 2005 䡠 VOLUME 105, NUMBER 9
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BLOOD, 1 MAY 2005 䡠 VOLUME 105, NUMBER 9
CYTOGENETIC-MOLECULAR CLASSIFICATION OF ADULT ALL
3435
Based on these considerations, the prospective Italian multicenter Gruppo Italiano Malattie Ematologiche dell’Adulto
(GIMEMA) 0496 study, started in 1996, provided for all adult
patients with ALL entering the protocol a multiparametric diagnostic approach including conventional cytogenetics and molecular
analysis for the following gene rearrangements: BCR-ABL, MLLAF4, E2A-PBX1, TEL-AML1, MLL, and p15 and p16 deletion. This
effort aimed at a careful characterization of the genetic lesions
occurring in adult ALL and at evaluating their prognostic significance in a large cohort of patients treated homogeneously.
cells with the same chromosomal loss, or at least 20 metaphases without clonal
changes. However, in less than 10% of cases classified as normal, 10 to 19 mitotic
figures could be analyzed. A central karyotype review was also performed in
difficult cases. Each patient was classified hierarchically in different subgroups
on the basis of the cytogenetic abnormality; in particular, karyotypic aberrations
with known adverse prognostic significance as t(9;22), t(4;11), t(1;19) took
precedence over others with less defined prognostic value, as del(6q) and del(9p).
If none of these was identified, patients having structural aberrations with or
without numerical changes not exceeding one chromosome gain or loss were
assigned to the miscellaneous group, whereas patients carrying numerical
aberrations only were assigned to the hyperdiploid group. Patients without
karyotypic anomalies were included in the normal group.
Patients, materials, and methods
DNA analysis
The GIMEMA ALL 0496 protocol, which began in October 1996 and was
closed in September 2000, contemplated a central handling and biologic
investigation of bone marrow (BM) or peripheral blood (PB) samples of the
enrolled patients at presentation including conventional cytogenetics,
Southern blot and reverse transcription-polymerase chain reaction (RTPCR), and multidrug resistance (MDR1) expression. Samples were sent by
overnight dispatch to the laboratories of the Department of Cellular
Biotechnologies and Hematology, University “La Sapienza” of Rome; the
specific investigations were performed at the same center, at the Department of Clinical and Biological Sciences, “S. L. Gonzaga” Hospital,
Orbassano, University of Turin, at the Hematology and Bone Marrow
Transplantation Unit, University of Perugia, at the CEINGE and Department of Biochemistry and Medical Biotechnologies, University “Federico
II” of Naples, and at the Department of Biomedical Science, Hematology
Unit, University of Ferrara, all in Italy.
All patients were treated according to the ALL 0496 protocol, which is
derived from the ALLVR589 regimen10 and included patients aged 16 to 60
years with a diagnosis of ALL, with the exclusion of L3 B-cell ALL
(B-ALL). The study was approved by the Institutional Review Board of the
Department of Cellular Biotechnologies and Hematology, University “La
Sapienza” of Rome. Informed consent was provided according to the
Declaration of Helsinki. The protocol design has been described elsewhere11; in summary it comprised an induction period including high-dose
daunorubicin, a consolidation with high-dose ara-C, chemoprophylaxis and
radioprophylaxis of central nervous system involvement, and periodic
reinductions over a 3-year maintenance period. Patients positive for
Philadelphia chromosome (Ph⫹) achieving complete remission (CR) were
treated according to the HAM protocol that included high-dose ara-C (3
g/m2/12 h for 4 days) and mitoxantrone (10 mg/m2/24 h for 3 days)
followed by allogeneic or autologous hemopoietic stem cell transplantation
based on the availability of an HLA-identical sibling.12
Cytology and immunology
The immunophenotype of ALL patients was performed by the referring
centers on BM or PB samples at diagnosis. The reactivity with monoclonal
antibodies directed against lymphoid and myeloid-associated antigens
(TdT, HLA-DR, CD19, CD10, CD20, CyIg, CD2, CD5, CD7; CD3, CD13,
CD33, CD14, CD34) was evaluated by flow cytometry. Fixed cytocentrifuge preparations were used for the detection of TdT and CyIg␮ chains by
immunocytochemical staining. The results were reviewed by the GIMEMA
biologic committee. The surface markers were considered positive when
20% or more of the blasts expressed the antigen; intracytoplasmic
positiveness was based on 10% or more reactive blasts. The immunologic
definition in B- and T-lineage ALL was based on the criteria defined by the
European Group for the Immunological Characterization of Leukemias.13
High-molecular-weight DNA was extracted from Ficoll-Hypaque–isolated
mononuclear cells, digested to completion with EcoRI, HindIII, or BglII
restriction endonucleases, size-fractionated by electrophoresis through
0.8% agarose gels, denatured, and transferred to nitrocellulose membranes.
These DNAs were prehybridized for 2 to 4 hours at 42°C in a solution
containing 50% deionized formamide, 5 ⫻ Denhardt solution, and 100
␮g/mL denatured salmon sperm and then hybridized at 42°C for 16 to 18
hours in the same solution with the denatured p16 exon 2 probe previously
labeled with 32P by the random priming technique.11 Filters were washed at
room temperature for 10 minutes in 0.2 ⫻ standard saline citrate/0.1%
sodium dodecyl sulfate and at 50°C for 15 to 20 minutes in the same
solution. Filters were then exposed for 2 to 8 days at ⫺80°C for
autoradiography using intensifying screens.
The B859 probe previously described, which explores the MLL
breakpoint region at chromosome 11q23, was also used to study the
configuration of this gene in ALL patients.15 In addition to the detection of
rearrangements at this locus, this probe served as control for the assessment
of the DNA amount present in the filters. The genomic deletions of the p15
and p16 genes were evaluated in 211 patients.
RT-PCR
Total RNA was extracted from BM or PB mononuclear cells collected at
diagnosis and cryopreserved in guanidinium isothiocyanate, according to
the method of Chomczynski and Sacchi.16 To identify the most frequent
fusion genes in ALL—namely, MLL-AF4, MLL-ENL, the BCR-ABL p190
(e2a2) and p210 (b2a2, b3a2) isoforms, E2A-PBX1, TEL-AML1—we used a
multiplex RT-PCR strategy17 in 60 cases and the standardized RT-PCR
protocol defined by the Biomed-1 Concerted Action in the remaining.18
Analyses were conducted in 3 referral centers (Rome, Turin, and Naples)
and centrally reviewed. Because the therapeutic program contemplated a
different postremission strategy for patients with BCR-ABL⫹ ALL, the
BCR-ABL fusion product detection was performed in all patients. The
presence of the MLL-AF4, E2A-PBX, and TEL-AML1 fusion transcripts was
investigated in 287, 162, and 162 cases, respectively.
MDR1 expression
MDR1 expression was assessed on BM or PB samples by depicting the
leukemic cells with the MRK16 antibody (Kamiya Biomedical, Seattle,
WA), which reacts with a cell surface epitope of the P-glycoprotein (P-gp)
170, as previously reported.19 Flow cytometry was used to measure the
fluorescence intensity of the leukemic samples and of the MDR1⫹ cell lines
used as controls in the analysis standardization. Differences in fluorescence
intensity were evaluated using the Kolmogorov-Smirnov statistic test (D).20
According to the study priority, 250 cases were analyzed in one referral
laboratory in Rome.
Cytogenetic analysis
Statistical analyses
Cytogenetic analyses were performed in 3 referral centers (Rome, Ferrara,
and Perugia) on direct and 24-hour cultured preparations of BM or PB cells
(or both) without stimulation. GTG-banding chromosomes were classified
according to the International System for Human Cytogenetic Nomenclature.14 A successful cytogenetic analysis required the detection of at least 2
or more cells with the same structural change or chromosomal gain, 3 or more
Comparison of pretreatment characteristics on CR rate was carried out
using the ␹2 test or Fisher exact test. The relationship of the outcome with
age, white blood cells (WBCs), phenotype, MDR, and cytogeneticmolecular classification was analyzed using the logistic and Cox regression
models.21 In the multivariate analysis, the classification in 2 cytogeneticmolecular risk groups was tested with the following variables: age and
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3436
BLOOD, 1 MAY 2005 䡠 VOLUME 105, NUMBER 9
MANCINI et al
Table 1. Distribution by cytogenetic hierarchical classification
Groups
No. cases
% of cases
Normal
114
30.2
del(9p)
2
0.5
del(6q)
15
4.0
Hyperdiploid
25
6.6
Miscellaneous structural abnormalities
41
10.8
t(9;22)
64
16.9
t(4;11)
18
4.8
t(1;19)
3
0.8
Failed
96
25.4
Total
378
100.0
WBC count as continuous variables; immunophenotype was dichotomized
as expression of B versus T markers; MDR expression was dichotomized as
present (⫽ 0.05) or absent. The probability of disease-free survival (DFS)
and overall survival (OS) was calculated using the Kaplan and Meier
method22 and the prognostic value of potential factors was analyzed using
the log-rank test with stratification for risk group.23
investigation (6 cases) or absence of molecular abnormalities in
cases with missing cytogenetic samples (22 cases). BCR-ABL
fusion was detected in 102 of 386 cases (26.4%; Table 2). Of
these, 58 (57%) showed a p190, 23 (23%) a p210, 19 (19%) a
p190/210 fusion protein, and 1 a rare e1a3 fusion. AF4-MLL,
E2A-PBX1, and TEL-AML1 fusion genes were identified in 23
(8%) of 287, 6 (3.7%) of 162, and 1 (0.6%) of 162 of the
investigated cases, respectively. A homozygous or hemizygous
loss of p15 or p16 genes (or both) was documented in 52
(24.6%) of 211 analyzed cases. Among the 52 deleted cases, 30
showed the deletion as an isolated anomaly. BCR-ABL (11
cases), E2A-PBX1 (1 case), and AF4-MLL (1 case) fusions
emerged as the molecular changes more frequently associated
with p15/p16 deletion. Of the 386 cases molecularly investigated, 61 with molecular analysis negative for the presence of
fusion genes or p15-p16 deletion and inadequate cytogenetic
results (no mitosis or ⬍ 10 analyzed cells with normal karyotype) were considered as not evaluable.
Integrated cytogenetic and molecular classification
Results
Cytogenetics
Conventional cytogenetics was performed on 378 of 414 cases
centrally processed, due to inadequate samples for cytogenetic
analysis (6 cases) or missing cytogenetic samples (30 cases). An
evaluable karyotype was obtained in 282 cases (75%) and an
abnormal clone was detected in 168 (60%). Cases with a defined
karyotype were classified in different subgroups following the
hierarchical approach indicated (Table 1). Among cases with
structural changes, the subgroup with the t(9;22) translocation was
the largest (64 of 378; 16.9%). In this group, additional chromosomal changes were identified in 55% of the cases and included an
additional Ph (8 cases), 9p deletion (3 cases), and chromosomal
gain resulting in hyperdiploidy (3 cases). Patients with t(9;22) had
B-lineage ALL in all cases but one (a T-ALL) with expression of
the CD34 and CD10 antigens in more than 90 of cases, a WBC
count more than 10 ⫻ 109/L in more than 60%, and an age older
than 30 years in 78% of cases. A t(4;11) translocation was identified
in 18 patients (4.8%); 5 had additional structural anomalies that
included i(7q) (1 case) and del(6q) (1 case). A t(1;19) translocation
was detected in 3 cases (0.8%); 2 had the unbalanced form and 1
the balanced form of the translocation; all cases showed additional
changes including in one instance a del(6q). Partial deletions of the
long arm of chromosome 6 were identified in 15 cases (4%); of
these, 1 had the presence of a BCR-ABL rearrangement at the molecular
level. A T-cell phenotype was found in 8 (57%) of 14 cases, and in 6 of
them the anomaly was present as isolated change. An isolated del(9p)
was identified in 2 cases, both showing a B-cell phenotype. The other 41
cases (10.8%) with an abnormal karyotype were combined to form the
miscellaneous group. The most common structural changes included in
this group were del(7q) (3 cases), del(5q) (2 cases), del(12p) or t(12p) (2
cases), t(10;14)(q24;q11) (2 cases), t(4;11)(q21;p15) (1 case), and 14q
abnormalities (5 cases). Numerical aberrations consisting in low hyperdiploidy (47-50 chromosomes, 16 cases) and in high hyperdiploidy
(⬎ 50 chromosomes, 9 cases) were identified in 25 patients (6.6%).
Finally, 114 cases (30.2%) without apparent structural or numerical
changes were recorded.
Molecular analysis
Molecular analyses were performed on 386 of 414 cases
centrally collected, owing to inadequate samples for molecular
Considering the cases having both conventional cytogenetics (282
cases) or molecular analysis (325 cases) successfully performed, a
total of 325 patients with a defined genetic profile were hierarchically classified in the following subgroups: normal, BCR-ABL/t(9;
22), MLL-AF4/t(4;11), E2A-PBX1/t(1;19), 6q deletions, miscellaneous, hyperdiploid, 9p and/or p15 and/or p16 deletions.
This integrated cytogenetic-molecular approach ensures a more
precise categorization of patients with respect to the presence or
absence of distinct genetic alterations.
The frequency of each cytogenetic-molecular subgroup and
their associated biologic and clinical features are presented in
Tables 3 and 4. With respect to previously cytogenetic-based
classifications, this classification system allowed us to delineate,
in particular, 3 subgroups of patients: namely, normal, 9p/p15p16 deletions, and hyperdiploid, as much as possible free from
confounding genetic abnormalities. Compared with the other
cytogenetic-molecular subgroups, patients included in the normal subgroup showed more frequently an age younger than 30
years (59.3% versus 40.6%; P ⫽ .003) and a WBC count lower
than 30 ⫻ 103/L (72.1% versus 56.1; P ⫽ .009). As for the
del(6q) subgroup, patients included in the group with isolated
cytogenetic or molecular 9p/p15-p16 abnormalities had more
frequently a T-cell lineage ALL than patients of the other
subgroups (57.1% versus 36.7%, P ⫽ ⬍ .001). The hyperdiploid group showed a CD10⫺ B-lineage ALL more frequently
than the other groups (58.3% versus 32.2%; P ⫽ .009) and
tended to have more frequently a WBC count lower than
30 ⫻ 103/L (76% versus 59%), as in the normal subgroup.
Treatment results
Of the 325 patients registered and eligible for the study who could
be considered for the integrated cytogenetic-molecular classification, 317 started the induction treatment and 294 were evaluable for
Table 2. Distribution by molecular abnormalities
Type of transcript or
gene rearrangement
No. abnormal cases/ no. analyzed
cases (%)
BCR-ABL
101/386 (26.2)
MLL-AF4
23/287 (8.0)
TEL-AML1
E2A-PBX1
p15/p16 deletion
1/162 (0.6)
6/162 (3.7)
52/211 (24.6)
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BLOOD, 1 MAY 2005 䡠 VOLUME 105, NUMBER 9
CYTOGENETIC-MOLECULAR CLASSIFICATION OF ADULT ALL
3437
Table 3. Distribution of 386 ALL cases according to conventional cytogenetic and cytogenetic-molecular classification
Cytogenetic-molecular groups, no.
Cytogenetic group
Normal
t(9;22)/
BCRABL
t(4;11)/
AF4MLL
t(1;19)/
E2A-PB
XI
del(6q)
Hyperdiploid
Miscellaneous
del(9p)/
p15p16
Not
evaluable
Total
Normal
86
4
3
2
0
0
1*
18
0
114
t(9;22)
0
64†
0
0
0
0
0
0
0
64
t(4;11)
0
0
18‡
0
0
0
0
0
0
18
t(1;19)
0
0
0
3‡
0
0
0
0
0
3
del(6q)
0
1
0
0
14
0
0
0
0
15
Hyperdiploid
0
0
0
0
0
25
0
0
0
25
Miscellaneous
0
2
2
1
0
0
36
0
0
41
del(9p)
0
0
0
0
0
0
0
2
0
2
Not evaluable
0
25
1
1
0
0
0
8
61
96
Not done
Total
0
6
0
0
0
0
0
2
0
8
86
102
24
7
14
25
37
30
61
386
*TEL-AML1.
†One case with positive fluorescence in situ hybridization and negative RT-PCR.
‡One case missing molecular analysis.
the cytogenetic-molecular classification did not differ from that of
the other patients enrolled in the trial (CR rate, 80%; DFS and OS at
5 years, 33% [95% CI, 27.7%-37.9%], and 33% [95% CI,
28.3%-37.3%], respectively).
response (Table 5). Of these, 232 patients (79%) obtained CR. The
overall DFS was 31% at 5 years (95% CI, 24.4%-37.0%; Figure 1).
The OS at 5 years was 31% (95% CI, 25.6%-36.3%; Figure 2). The
overall clinical outcome of the patient population considered for
Table 4. Pretreatment characteristics of 325 genetically defined patients by cytogenetic-molecular groups
Cytogenetic-molecular groups
Total
Total
Normal
t(9;22)/
BCR-ABL
325
86
102
t(4;11)/
AF4-MLL
t(1;19)/
E2A-PBX1
del(6q)
del(9p)/
p15-p16
Pseudodiploid
24
7
14
30
37
Hyperdiploid
25
Sex, no. (%)
Male
194 (60)
51 (59)
54 (53)
13 (54)
3 (43)
11 (78)
24 (80)
22 (59)
Female
131 (40)
35 (41)
48 (47)
11 (46)
4 (57)
3 (22)
6 (20)
15 (41)
26 (15-58)
42 (16-60)
39 (15-59)
Median age, y (range)
31 (14-60)
22 (15-58)
27 (18-57)
16 (61)
9 (39)
29 (14-59)
23 (15-59)
22 (15-53)
Age, no. (%)
Younger than 30 y
148 (46)
51 (59)
22 (22)
7 (29)
6 (86)
8 (57)
15 (50)
25 (68)
14 (56)
31 to 50 y
124 (38)
29 (34)
51 (50)
14 (58)
0 (0)
5 (36)
10 (33)
7 (19)
8 (32)
51 to 60 y
53 (16)
6 (7)
29 (28)
3 (13)
1 (14)
1 (7)
5 (17)
5 (13)
3 (12)
WBC counts, no. (%)
10 ⫻ 109/L or less
132 (41)
49 (57)
36 (35)
3 (12.5)
3 (43)
4 (29)
10 (33)
14 (38)
13 (52)
10 ⫻ 109/L to 30 ⫻ 109/L
64 (20)
13 (15)
17 (17)
5 (21)
3 (43)
4 (29)
7 (23)
9 (24)
6 (24)
30 ⫻ 109/L to 50 ⫻ 109/L
32 (10)
8 (9)
12 (12)
3 (12.5)
0
2 (14)
3 (10)
2 (5)
2 (8)
50 ⫻ 109/L to 100 ⫻ 109/L
43 (13)
6 (7)
19 (19)
5 (21)
0
1 (7)
4 (14)
7 (19)
1 (4)
100 ⫻ 109/L or more
53 (16)
10 (12)
17 (17)
8 (33)
1 (14)
3 (21)
6 (20)
5 (14)
3 (12)
B
266 (82)
66 (77)
101 (99)
24 (100)
7 (100)
6 (43)
19 (63)
25 (68)
18 (72)
T
59 (18)
20 (23)
1 (1)
0
8 (57)
11 (37)
12 (32)
7 (28)
Phenotype, no. (%)
0
CD10 status, no. (%)
Positive
206 (66)
52 (64)
92 (93)
0 (0)
Negative
107 (34)
29 (36)
7 (7)
24 (100)
Not done
12
5
3
0
7 (100)
6 (46)
19 (63)
20 (57)
10 (42)
0 (0)
7 (54)
11 (37)
15 (43)
14 (58)
0
1
0
2
1
CD13 status, no. (%)
Positive
77 (26)
23 (29)
33 (35)
0 (0)
Negative
222 (74)
55 (71)
62 (65)
24 (100)
Not done
26
8
7
0
0 (0)
6 (100)
1
2 (15)
1 (4)
10 (29)
8 (38)
11 (85)
26 (96)
25 (71)
13 (62)
1
3
2
4
CD33 status, no. (%)
Positive
67 (22)
19 (24)
33 (35)
0 (0)
Negative
235 (78)
59 (76)
62 (65)
23 (100)
Not done
23
8
7
1
0 (0)
7 (100)
0
0 (0)
2 (7)
7 (20)
6 (26)
13 (100)
26 (93)
28 (80)
17 (74)
1
2
2
2
CD34 status, no. (%)
Positive
223 (72)
57 (71)
92 (95)
10 (41)
0 (0)
8 (57)
14 (48)
24 (76)
18 (75)
Negative
86 (28)
23 (29)
5 (5)
14 (59)
7 (100)
6 (43)
15 (52)
10 (24)
6 (25)
Not done
16
6
5
0
0
0
1
3
1
MDR, no. (%)
Yes
57 (23)
15 (24)
16 (21)
2 (11)
1 (14)
1 (9)
7 (26)
8 (27)
7 (37)
No
193 (87)
47 (76)
59 (79)
17 (89)
6 (86)
10 (91)
20 (74)
22 (73)
12 (63)
Not done
75
24
27
5
0
3
3
7
6
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3438
BLOOD, 1 MAY 2005 䡠 VOLUME 105, NUMBER 9
MANCINI et al
MDR1 expression
The MDR protein expression was assessed by MRK16 binding. MDR1 expression was detected in 57 (23%) of the 250
analyzed cases.
Prognostic value of the integrated molecular-cytogenetic
classification in ALL patients at presentation
Excluding patients not evaluable for response to induction, 294
of 325 cases could be assessed for their clinical outcome. No
significant differences among the 8 cytogenetic-molecular subgroups were observed in terms of CR rate (P ⫽ .238). In
contrast, on the basis of the median DFS, 3 prognostic groups
could be distinguished by the presence of specific cytogeneticmolecular abnormalities. Cases with normal karyotype and
isolated 9p/p15-p16 deletions are suggestive of having a relatively better outcome (median DFS, ⬎ 3 years) and were
considered as a “standard-risk” group, whereas patients with
t(9;22), t(4;11), and t(1;19) showed an adverse prognosis
(median DFS, 7 months) and were comprised in an “adverserisk” category. Patients with del(6q), miscellaneous abnormalities, and hyperdiploidies exhibited an intermediate outcome
(median DFS, 19 months) and were included in the “intermediaterisk” group. In univariate analysis, with respect to the DFS
probability at 2 years, patient stratification on the basis of
cytogenetic-molecular features was significantly associated with
outcome (P ⬍ .001; Figure 3). In a multivariate analysis model
including age, WBC count, MDR, and immunophenotype,
patients in the “high-risk” group had a statistically significant
lower probability to be alive without disease recurrence after 2
years from remission, compared to the “intermediate-risk”
(hazard ratio [HR] 2.00; P ⫽ .02), and patients in the “standardrisk” showed a significantly higher probability of being alive in
remission compared to those in the intermediate-risk group (HR
0.55; P ⫽ .04; Table 6). After a more prolonged follow-up, the
differences in outcome between the favorable and intermediate
groups tend to erode, becoming comparable at 5 years (Figure 3
and Tables 7-9).
Discussion
Despite the efforts to identify at the onset of the disease subgroups
of adult patients with ALL with a different prognostic likelihood,
the predictive value of the risk models so far proposed is still
limited. Considering that cytogenetic and molecular features are
among the most important factors for predicting outcome, we
hereby propose a risk classification system based on an integrated
cytogenetic-molecular characterization of a large series of patients
with adult ALL enrolled in the GIMEMA ALL 0496 trial and
studied prospectively at presentation. This systematic approach
first allowed us to decrease the number of cases with failed
cytogenetics (from 25% to 16%) or normal karyotype (from 29.5%
Figure 1. DFS of 232 patients obtained CR and considered in the integrated
cytogenetic-molecular classification.
to 22%) through the detection of molecular lesions not identified by
conventional cytogenetics and vice versa, and second, to depict 8
different cytogenetic-molecular subgroups as much as possible
homogeneous for defined genetic abnormalities, such as BCR-ABL
or t(9;22), MLL-AF4 or t(4;11), p15 and/or p16 deletion or del(9p),
and so forth (Table 5). Within the normal karyotype group, 28 of
114 patients had molecular anomalies missed by conventional
cytogenetics. The most frequently detected were p15 and p16
deletions (18 of 28 cases), t(9;22) (4 of 28 cases), and t(4;11) (3 of
28 cases). This confirms previous observations that most of the p15
and p16 deletions are not detected by conventional cytogenetics.24,25 In the group with failed cytogenetic analysis, 35 of 96 cases
were found to harbor molecular abnormalities consisting of BCRABL (25 of 35), AF4-MLL (1 of 35), and E2A-PBX1 (1 of 35)
rearrangements, and p15 or p16 deletions (8 of 35). Considering the
patients with normal or not evaluable cytogenetics, most molecular
abnormalities are restricted to the failed cytogenetic group (77.4%
versus 22.6%; P ⫽ ⬍ .001).
When the DFS probability at 2 years is considered as an early
prognostic end point, the cytogenetic-molecular subgroups delineated in
the present study allow prediction of a different clinical outcome
likelihood. Cases with no abnormalities or with 9p/p15 and/or p16
deletions have a significantly higher DFS probability, as shown in
Figure 3. The prognostic significance of a normal karyotype has been
seldom estimated in adult ALL and varies in different series. The results
of our study are consistent with the outcome of a series of 443 adult
Table 5. Flow chart of the patients included in the GIMEMA
0496 protocol
Clinical
trial
Evaluable integrated
Central cytogenetic-molecular
handling
classification
No. patients eligible for the study
498
414
No. patients who started induction
490
408
317
No. patients evaluable for response
455
380
294
363 (80)
303 (80)
232 (79)
No. RC (%)
325
Figure 2. OS of 325 patients considered for the integrated cytogeneticmolecular classification.
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BLOOD, 1 MAY 2005 䡠 VOLUME 105, NUMBER 9
Figure 3. DFS by univariate analysis. DFS according to (A) cytogenetic subgroups,
(B) cytogenetic-molecular subgroups, and (C) cytogenetic-molecular risk groups.
patients reported by the Groupe Français de Cytogénétique Hématologique (GFCH),3 which found the normal karyotype group to be
associated with a better prognosis (median event-free survival [EFS], 24
months; EFS 44%) than the abnormal karyotype group (P ⬍ .001) in
B-lineage ALL. This finding was not confirmed by the Medical
Research Council (MRC) UKALL XA trial,4 which showed that DFS
for patients with a diploid karyotype was comparable with the pooroutcome categories. This result, however, could be due to the presence
of poor-risk genetic lesions missed by conventional cytogenetics,
because in this study molecular investigations were not performed. The
frequency of normal karyotypes has been reported to range between
16% and 34% for adult ALL patients; clinical features associated with
this subgroup include a median age ranging between 30 and 44 years, a
median WBC count between 10 and 30 ⫻ 109/L, and a T-cell phenotype
in 18% to 38% of cases.1-5,26 In the present series, the subgroup with
normal karyotype represented 27% of cases and showed more frequently than the abnormal cytogenetic-molecular subgroups a young
CYTOGENETIC-MOLECULAR CLASSIFICATION OF ADULT ALL
3439
age (27 years; range, 14-58 years), a low WBC count (6.8 ⫻ 109/L;
range, 0.6-700 ⫻ 109/L), whereas the prevalence of T-cell phenotype
was similar to that of the other groups (23%).
In addition to normal karyotype, the standard-risk group
included cases carrying abnormalities of the short arm of chromosome 9, including p15 and p16 deletions. These abnormalities have
been found in approximately 10% of childhood ALL with a higher
percentage in T-ALL and in 5% to 16% of adults.27-31 Frequently,
9p deletions have been associated with other abnormalities, in most
cases the t(9;22) translocation, and are considered a secondary
change.5,24,30 In our series, 9p, p15 and/or p16 deletions were found
in 52 (25%) of 211 analyzed cases emerging as the most frequent
molecular abnormalities after BCR-ABL. In 30 cases, 19 with Band 11 with T-cell phenotype, this group of anomalies was present
as an isolated change, suggesting that it may be considered as a
primary change in ALL. Comparing this group with the 22 cases
carrying 9p abnormalities in addition to other changes consisting of
a t(9;22) (11 cases), other structural changes included in the
pseudodiploid subgroup (7 cases), a del(6q) (2 cases), a t(1;19) (1
case), and a t(4;11) (1 case), patients with isolated 9p/p15 and/or
p16 deletion showed a similar median age (27 versus 29 years), but
a lower median WBC count (21 versus 46 ⫻ 109/L). In a pediatric
series treated with a Children’s Cancer Group risk-adjusted protocol, Heerema et al27 reported that deletions of 9p were associated
with an adverse prognosis in B-lineage ALL and, in the homozygous form, also in T-ALL. This finding has been confirmed by
Carter et al,32 who reported that homozygous and hemizygous
deletions of p16 are independent predictors of relapse. Disagreement still exists about the correlation with prognostic and immunophenotypic features of 9p abnormalities in adult ALL. Fizzotti et
al33 found a significant relationship between p16 deletion and a
subset of ALL with features of aggressive disease (high leukemic
cell mass and high WBC counts), but no differences by immunophenotype. Indeed, the GFCH reported no impact on prognosis and no
significant correlation with B- or T-lineage ALL.3 Similar findings
were observed by Faderl et al,24 who reported no difference in the
outcome of patients with homozygous p15 and p16 gene deletions
with respect to patients with no deletions or loss of only one allele.
The correlation of 9p abnormalities with an adverse prognosis may
depend on their frequent association with additional karyotypic
changes, often consisting in poor-risk aberrations such as t(9;22),
t(4;11), or complex abnormalities. Conversely, our study suggests
that the p15 and/or p16 deletions, when present as an isolated
change, are not associated with a high risk of relapse at short term.
The intermediate prognostic category included patients with
del(6q), cases with diploid karyotype, and a miscellaneous group of
other structural changes not encompassed by the other 2 risk
groups, which were too infrequent to be assigned a prognostic
significance in their own right, as well as cases with only numerical
acquisitions. Partial deletions of the long arm of chromosome 6
Table 6. Multivariate analysis for DFS at 2 years
Variable
P value
Hazard
ratio
95% hazard ratio
confidence limits
WBCs, ⫻ 109/L*
0.0479
1.002
1.000
1.003
Age, y†
0.2061
1.010
0.994
1.026
Phenotype, B or T
0.2924
1.385
0.755
2.538
MDR, negative or positive
0.7815
0.928
0.546
1.575
Intermediate vs standard
0.0428
0.552
0.311
0.981
Intermediate vs high
0.0231
2.006
1.100
3.659
Cytogenetic-molecular risk group
*Range, 0.5-848 ⫻
†Range, 16-60 years.
109/L.
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3440
BLOOD, 1 MAY 2005 䡠 VOLUME 105, NUMBER 9
MANCINI et al
Table 7. Univariate analysis for DSF according to
cytogenetic subgroups
ⴙ, y
Table 9. Univariate analysis for DSF according to cytogeneticmolecular risk groups
ⴙ, y
Median, % 95% CI, y No. patients No. events
95% CI, y
No. patients
No. events
del(9p)
Not reached*
—
—
2
0
Standard
3.2
38.6-60.6
85
47
Normal
2.8
49
38.2-60.2
83
48
Intermediate
1.6
36.9-63.1
56
34
Hyperdiploid
1.6
50
28.1-71.9
20
12
High
0.62
39.2-59.7
91
75
del(6q)
1.6
45
16.0-74.9
11
8
Miscellaneous
1.3
48
30.1-66.5
29
17
t(1;19)
0.8
33
0-86.7
3
2
t(9;22)
0.6
50
34.1-65.9
38
34
t(4;11)
0.4
50
25.5-74.5
16
12
*del(9p) 100% at 4 years.
have mainly been reported in pediatric T-ALL, with a frequency
ranging between 6% and 10% of patients, and an association with a
favorable prognosis. In adults, too few cases have been reported so
far to allow definitive clinical and biologic correlations. Our study
highlights the preferential correlation of this anomaly with a T-cell
phenotype, especially when it is present as isolated change, as
reported elsewhere.11 Moreover, the result of our analysis makes
previous not unfavorable prognostic suggestions somewhat questionable, on the basis of the median DFS of 19 months recorded in
the del(6q) subgroup. The hyperdiploid group, which includes 9
cases (32%) with so-called “high” hyperdiploidy (⬎ 51 chromosomes), accounts for 8% of successfully investigated cases, in line
with what so far is described in adult ALL. More than 50% of the
hyperdiploid cases described in the literature were of B-cell
phenotype, were younger than 30 years, and a normal WBC
count.3,4 The clinical outcome of patients with hyperdiploidy varies
in different series, being more favorable in children than in adults,
where a poor outcome has been repeatedly reported.3,5,26 Similarly,
in the present study a median DFS of 19 months has been recorded
in the hyperdiploid group, despite the absence of unfavorable
structural changes, indicating that adults may not enjoy the good
prognosis reported in children. Adverse risk features, which may
correlate with an unfavorable outcome, such as WBC count more
than 30 ⫻ 109/L and age older than 30 years,34,35 were observed in
this group in 24% and 44% of cases, respectively.
The t(9;22), t(4;11), and t(1;19) translocations defined a cytogenetic-molecular group with unfavorable outcome. With respect to
the first 2 abnormalities, our data are consistent with what is
reported by several groups in terms of poor outcome.3-5 The
association with a poor prognosis of the t(1;19) translocation,
which in this series was found in 4% of cases, is still debated. In
pediatric ALL, where this aberration is more common (8%),
improved outcome has been obtained with a more intensified
treatment.36 In adults, the GFCH3 described 11 cases, of which 9
failed therapy within 12 months from diagnosis; conversely, the
MRC UKALL XA4 did not confirm the adverse outcome, reporting
a 3-year DFS of 54% in 10 patients, 4 of them undergoing bone
marrow transplantation. In the present study, t(1;19) characterizes a
Table 8. Univariate analysis for DSF according to cytogeneticmolecular subgroups
ⴙ, y
Median, %
95% CI, y
No. patients
No. events
del(9p)/p15-p16
4.0
45
22.0-68.0
22
12
Normal
2.9
50
37.7-63.1
63
35
Hyperdiploid
1.6
50
28.1-71.9
20
12
del(6q)
1.6
50
19.0-81.0
10
7
Pseudodiploid
1.3
50
30.8-69.2
26
15
t(1;19)/E2A-PBX1
0.6
43
6.2-79.5
7
5
t(9;22)/BCR-ABL
0.6
49
36.9-61.6
63
53
t(4;11)/AF4-MLL
0.4
48
26.3-69.0
21
17
subset of relatively young patients with CD10⫹, often CyIg⫹,
B-lineage ALL who do not express CD34 and myeloid associated
antigens. Despite the high CR rate, this anomaly was associated
with early treatment failure in 5/7 patients.37
Our study emphasizes the feasibility of a combined cytogeneticmolecular classification of adult patients with ALL in the context of
a multicenter clinical trial. At 2 years, the stratification in 3 risk
groups derived from this approach emerges, in univariate and
multivariate analysis, as a variable significantly associated with
DFS probability. Although at 5 years the favorable and intermediate groups had an overall comparable outcome, this model
highlights a category of patients with at least 2 years of relatively
favorable outcome during which, on the basis of the presence of
minimal residual disease, the impact of consolidative treatment
strategies including newer drugs should be evaluated.
In conclusion, our data indicate that a combined broad cytogeneticmolecular classification of adults with ALL at presentation provides a
framework that clearly distinguishes groups of patients with a different
likelihood of relapse and that, in the context of multicenter protocols,
this integrated approach is essential for the design of a risk-adapted
therapeutic algorithm. The challenge of future trials will be to further
extend the number of cytogenetic-molecular profiled cases by using
novel techniques, such as spectral karyotyping and comparative genomic
hybridization, as well as to complement the genetic screening with other
parameters in order, ultimately, to permit a more refined and targeted
clinical management.
Acknowledgments
The authors thank Francesca Paoloni for biostatistical analysis and
Sandra De Simone for data management.
Appendix
The following institutions and investigators participated in this study:
Ospedale SG Moscati, Avellino, Ettore Volpe; Istituto di Ematologia
L. A. Seràgnoli, Bologna, Michele Baccarani; Ospedale S Giovanni
Bosco, Napoli, Eustachio Miraglia; Ospedale S. Francesco, Attilio
Gabbas; Ospedale S. Carlo, Potenza, Francesco Ricciuti; Università
Cattolica, Roma, Giuseppe Leone; Ospedale Cardarelli, Napoli, Vincenzo Mettivier; Ospedale S Giovanni Battista, Torino, Mario Boccadoro; Ospedale SS Antonio e Biagio, Alessandria, Alessandro Levis;
Ospedale S Maria Goretti, Latina, Angelo De Blasio; Università di
Palermo, Pietro Citarrella; Ospedale S Antonio Abate, Gallarate,
Ruggero Mozzana; Arcispedale S Maria Nuova, Reggio Emilia, Luigi
Gugliotta; Ospedale S Eugenio, Roma, Sergio Amadori; Università di
Sassari, Maurizio Longinotti; Ospedale S Martino, Genova, Gino
Santini; Dipartimento di Medicina Interna, Università di Genova,
Riccardo Ghio; Ospedale A Di Summa Brindisi, Giovanni Quarta;
Ospedale A Sclavo, Siena, Francesco Lauria; Ospedale Maggiore, Lodi,
Giulio Nalli; Ospedale E Morelli, Sondalo, Renzo Epis; CRO Aviano,
Umberto Tirelli; Università di Palermo, Vincenzo Abbadessa; Ospedale
S Salvatore, Pesaro, Giuseppe Visani; Ospedale S Croce e Carle, Cuneo,
Andrea Gallamini; Ospedale SS Annunziata, Taranto, Patrizio Mazza;
Ospedale Maggiore della Carità, Novara, Giancarlo Avanzi.
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BLOOD, 1 MAY 2005 䡠 VOLUME 105, NUMBER 9
CYTOGENETIC-MOLECULAR CLASSIFICATION OF ADULT ALL
3441
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2005 105: 3434-3441
doi:10.1182/blood-2004-07-2922 originally published online
January 13, 2005
A comprehensive genetic classification of adult acute lymphoblastic
leukemia (ALL): analysis of the GIMEMA 0496 protocol
Marco Mancini, Daniela Scappaticci, Giuseppe Cimino, Mauro Nanni, Valentina Derme, Loredana
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